Embedding of HIV Egress within Cortical F-Actin

F-Actin remodeling is important for the spread of HIV via cell–cell contacts; however, the mechanisms by which HIV corrupts the actin cytoskeleton are poorly understood. Through live cell imaging and focused ion beam scanning electron microscopy (FIB-SEM), we observed F-Actin structures that exhibit strong positive curvature to be enriched for HIV buds. Virion proteomics, gene silencing, and viral mutagenesis supported a Cdc42-IQGAP1-Arp2/3 pathway as the primary intersection of HIV budding, membrane curvature and F-Actin regulation. Whilst HIV egress activated the Cdc42-Arp2/3 filopodial pathway, this came at the expense of cell-free viral release. Importantly, release could be rescued by cell–cell contact, provided Cdc42 and IQGAP1 were present. From these observations, we conclude that a proportion out-going HIV has corrupted a central F-Actin node that enables initial coupling of HIV buds to cortical F-Actin to place HIV at the leading cell edge. Whilst this initially prevents particle release, the maturation of cell–cell contacts signals back to this F-Actin node to enable viral release & subsequent infection of the contacting cell.

The Role of L-Selectin in HIV Infection

HIV envelope glycoprotein is the most heavily glycosylated viral protein complex identified with over 20 glycans on its surface. This glycan canopy is thought to primarily shield the virus from host immune recognition as glycans are poor immunogens in general, however rare HIV neutralizing antibodies nevertheless potently recognize the glycan epitopes. While CD4 and chemokine receptors have been known as viral entry receptor and coreceptor, for many years the role of viral glycans in HIV entry was controversial. Recently, we showed that HIV envelope glycan binds to L-selectin in solution and on CD4 T lymphocytes. The viral glycan and L-selectin interaction functions to facilitate the viral adhesion and entry. Upon entry, infected CD4 T lymphocytes are stimulated to progressively shed L-selectin and suppressing this lectin receptor shedding greatly reduced HIV viral release and caused aggregation of diminutive virus-like particles within experimental infections and from infected primary T lymphocytes derived from both viremic and aviremic individuals. As shedding of L-selectin is mediated by ADAM metalloproteinases downstream of host-cell stimulation, these findings showed a novel mechanism for HIV viral release and offer a potential new class of anti-HIV compounds.

Release of infectious virus and cytokines in nasopharyngeal swabs from individuals infected with non-B.1.1.7 or B.1.1.7 SARS-CoV-2 variants.

The mechanisms that allowed for the SARS-CoV-2 B.1.1.7 variant to rapidly outcompete pre-existing variants in many countries remain poorly characterized. Here, we analyzed viral release, anti-SARS-CoV-2 antibodies and cytokine production in a retrospective series of 427 RTqPCR+ nasopharyngeal swabs collected in COVID-19 patients harbouring either non-B.1.1.7 or B.1.17 variants. We utilized a novel rapid assay, based on S-Fuse-T reporter cells, to quantify infectious SARS-CoV-2. With both non-B.1.1.7 and B.1.1.7 variants, viral titers were highly variable, ranging from 0 to >106 infectious units, and correlated with viral RNA levels. Lateral flow antigenic rapid diagnostic tests (RDTs) were positive in 96% of the samples harbouring infectious virus. About 67 % of individuals carried detectable infectious virus within the first two days after onset of symptoms. This proportion decreased overtime, and viable virus was detected up to 14 days. Samples containing anti-SARS-CoV-2 IgG or IgA did not generally harbour infectious virus. The proportion of individuals displaying viable virus or being RDT-positive was not higher with B.1.1.7 than with non- B.1.1.7 variants. Ct values were slightly but not significantly lower with B.1.1.7. The variant was characterized by a fast decrease of infectivity overtime and a marked release of 17 cytokines (including IFN-b, IP-10, IL-10 and TRAIL). Our results highlight differences between non-B.1.1.7 and B.1.1.7 variants. B.1.1.7 is associated with modified viral decays and cytokine profiles at the nasopharyngeal mucosae during symptomatic infection.

The E3 Ubiquitin Ligase RNF5 Facilitates SARS-CoV-2 Membrane Protein-Mediated Virion Release

As enveloped virus, SARS-CoV-2 membrane protein (M) mediates viral release from cellular membranes, but the molecular mechanisms of SARS-CoV-2 virions release remain poorly understood. Here, we performed RNAi screening and identified the E3 ligase RNF5 which mediates ubiquitination of SARS-CoV-2 M at residue K15 to enhance the interaction of viral envelope (E) with M. M-E complex ensures the uniform size of viral particles for viral maturation and mediates viral release. Moreover, overexpression of M induces complete autophagy which is dependent on RNF5-mediated ubiquitin modification. M inhibits the activity of lysosome protease, and uses autolysosomes for virion release. Consequently, all these results demonstrate that RNF5 mediates ubiquitin modification of SARS-CoV-2 M to stabilize the M-E complex and induce autophagy for virion release.

Viral Release Threshold in the Salivary Gland of Leafhopper Vector Mediates the Intermittent Transmission of Rice Dwarf Virus

Numerous piercing-sucking insects can persistently transmit viral pathogens in combination with saliva to plant phloem in an intermittent pattern. Insect vectors maintain viruliferous for life. However, the reason why insect vectors discontinuously transmit the virus remains unclear. Rice dwarf virus (RDV), a plant reovirus, was found to replicate and assemble the progeny virions in salivary gland cells of the leafhopper vector. We observed that the RDV virions moved into saliva-stored cavities in the salivary glands of leafhopper vectors via an exocytosis-like mechanism, facilitating the viral horizontal transmission to plant hosts during the feeding of leafhoppers. Interestingly, the levels of viral accumulation in the salivary glands of leafhoppers during the transmitting period were significantly lower than those of viruliferous individuals during the intermittent period. A putative viral release threshold, which was close to 1.79 × 104 copies/μg RNA was proposed from the viral titers in the salivary glands of 52 leafhoppers during the intermittent period. Thus, the viral release threshold was hypothesized to mediate the intermittent release of RDV from the salivary gland cells of leafhoppers. We anticipate that viral release threshold-mediated intermittent transmission by insect vectors is the conserved strategy for the epidemic and persistence of vector-borne viruses in nature.

Reinfection with SARS-CoV-2 in a splenectomized patient from Kocaeli, Turkey

Background: The COVID-19 pandemic has resulted in more than 31 million cases diagnosed and 965,000 deaths as of September 21, 2020. As the number of cases increases, clinical conditions such as prolonged viral release, reinfection, and reactivation are being encountered more frequently.Case presentation: This article presents a case who was reinfected with COVID-19 and developed a more severe second disease unlike other cases in the literature.Conclusions: Reinfection in a splenectomy case may help researchers understand the pathogenesis of the disease and can be a guide for the development of vaccines and new treatments.

In Support of Simian Polyomavirus 40 VP4 as a Later Expressed Viroporin

ABSTRACT Simian virus 40 VP4 was discovered in 2007 as a later expressed viral protein initiated from a downstream Met on the VP2/VP3 transcript. VP4’s role as a viroporin involved in viral release was supported in a series of additional articles that characterized the ability of VP4 to associate with and permeabilize biological membranes. This commentary is our response to the perspective from Henriksen and Rinaldo (mSphere 5:e00019-20, 2020, https://doi.org/10.1128/mSphere.00019-20) that challenges the existence of SV40 VP4.

HIV corrupts the Cdc42-IQGAP1 axis of actin regulation to promote cell-cell viral spread

F-Actin remodelling is important for the spread of HIV via cell-cell contacts, yet the mechanisms by which HIV corrupts the actin cytoskeleton are poorly understood. Through live cell imaging and focused ion beam scanning electron microscopy (FIB-SEM), we observed F-Actin structures that exhibit strong positive curvature to be enriched for HIV buds. Virion proteomics, gene silencing, and viral mutagenesis supported a Cdc42-IQGAP1-Arp2/3 pathway as the primary intersection of HIV budding, membrane curvature and F-Actin regulation. Whilst HIV egress activated the Cdc42-Arp2/3 filopodial pathway, this came at the expense of cell-free viral release. Importantly, release could be rescued by cell-cell contact, providing Cdc42 and IQGAP1 were present. From these observations we conclude that out-going HIV has corrupted a central F-Actin node that enables initial coupling of HIV buds to cortical F-Actin to place HIV at the leading cell edge. Whilst this initially prevents particle release, maturation of cell-cell contacts signals back to this F-Actin node to enable viral release & subsequent infection of the contacting cell.

Upregulation of BST-2 by Type I Interferons Reduces the Capacity of Vpu To Protect HIV-1-Infected Cells from NK Cell Responses

ABSTRACTThe HIV-1 accessory protein Vpu enhances viral release by counteracting the restriction factor BST-2. Furthermore, Vpu promotes NK cell evasion by downmodulating cell surface NTB-A and PVR, known ligands of the NK cell receptors NTB-A and DNAM-1, respectively. While it has been established that Vpu’s transmembrane domain (TMD) is required for the interaction and intracellular sequestration of BST-2, NTB-A, and PVR, it remains unclear how Vpu manages to target these proteins simultaneously. In this study, we show that upon upregulation, BST-2 is preferentially downregulated by Vpu over its other TMD substrates. We found that type I interferon (IFN)-mediated BST-2 upregulation greatly impairs the ability of Vpu to downregulate NTB-A and PVR. Our results suggest that occupation of Vpu by BST-2 affects its ability to downregulate other TMD substrates. Accordingly, knockdown of BST-2 increases Vpu’s potency to downmodulate NTB-A and PVR in the presence of type I IFN treatment. Moreover, we show that expression of human BST-2, but not that of the macaque orthologue, decreases Vpu’s capacity to downregulate NTB-A. Importantly, we show that type I IFNs efficiently sensitize HIV-1-infected cells to NTB-A- and DNAM-1-mediated direct and antibody-dependent NK cell responses. Altogether, our results reveal that type I IFNs decrease Vpu’s polyfunctionality, thus reducing its capacity to protect HIV-1-infected cells from NK cell responses.IMPORTANCEThe restriction factor BST-2 and the NK cell ligands NTB-A and PVR are among a growing list of membrane proteins found to be downregulated by HIV-1 Vpu. BST-2 antagonism enhances viral release, while NTB-A and PVR downmodulation contributes to NK cell evasion. However, it remains unclear how Vpu can target multiple cellular factors simultaneously. Here we provide evidence that under physiological conditions, BST-2 is preferentially targeted by Vpu over NTB-A and PVR. Specifically, we show that type I IFNs decrease Vpu’s polyfunctionality by upregulating BST-2, thus reducing its capacity to protect HIV-1-infected cells from NK cell responses. This indicates that there is a hierarchy of Vpu substrates upon IFN treatment, revealing that for the virus, targeting BST-2 as part of its resistance to IFN takes precedence over evading NK cell responses. This reveals a potential weakness in HIV-1’s immunoevasion mechanisms that may be exploited therapeutically to harness NK cell responses against HIV-1.

Dendritic cells mature to resist lamin degradation and herpes virus release

Herpes simplex viruses bud into the nuclear membrane of infected cells. Turan et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201801151) demonstrate that mature dendritic cells control the peripheral location of lysosomes, reducing autophagic degradation of lamins and inhibiting viral release.